Inbred watermelon line WDL0090

ABSTRACT

One embodiment relates to seed and plants of inbred watermelon line WDL0090. Another embodiment relates to the plants, seeds and tissue cultures of inbred watermelon line WDL0090, and to methods for producing a watermelon plant produced by crossing such plants with themselves, with another watermelon plant, such as a plant of another genotype, or with vegetatively propagating said plant. Another embodiment further relates to seeds and plants produced by such crossing. Further embodiments relate to parts of such plants, including the fruit and gametes of such plants.

BACKGROUND

Citrullus lanatus, watermelon, is a plant which is included in thefamily Cucurbitaceae. Watermelon is an economically important crop andis used in a number of consumer and commodity products.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY

It is to be understood that the embodiments include a variety ofdifferent versions or embodiments, and this summary is not meant to belimiting or all-inclusive. This summary provides some generaldescriptions of some of the embodiments, but may also include some morespecific descriptions of other embodiments.

The embodiments recited herein relates to a novel and distinct inbredwatermelon, C. lanatus, line designated WDL0090, and to the seeds, plantparts, and tissue culture produced by that inbred watermelon line. Theembodiments further relate to fresh food and food products produced frominbred watermelon line WDL0090, such as, but not limited to productssuch as watermelon fruit, powders, juices, sweetener, colorings,watermelon pulp, watermelon shreds, raffinate and betaine. Allpublications cited in this application are herein incorporated byreference.

An embodiment provides an inbred watermelon line designated WDL0090.Another embodiment relates to the seeds of inbred watermelon lineWDL0090, to the plants of inbred watermelon line WDL0090 and to methodsfor producing a watermelon plant produced by crossing inbred watermelonline WDL0090 with itself or another inbred watermelon line, and thecreation of variants by mutagenesis or transformation of inbredwatermelon line WDL0090.

Any such methods using inbred watermelon line WDL0090 are a furtherembodiment: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using inbred watermelonline WDL0090 as at least one parent are within the scope of theembodiments. Advantageously, inbred watermelon line WDL0090 could beused in crosses with other, different watermelon plants to produce firstgeneration (F₁) watermelon hybrid seeds and plants with superiorcharacteristics.

Another embodiment provides for single or multiple gene converted plantsof inbred watermelon line WDL0090. The transferred gene(s) may be adominant or recessive allele. The transferred gene(s) may confer suchtraits as herbicide tolerance, insect tolerance, tolerance forbacterial, fungal, or viral disease, male fertility, male sterility,enhanced nutritional quality, environmental stress tolerance, modifiedcarbohydrate metabolism, modified yield, modified glycoalkaloid content,and industrial usage. The gene may be a naturally occurring watermelongene or a transgene introduced through genetic engineering techniques.

Another embodiment provides for regenerable cells for use in tissueculture of inbred watermelon line WDL0090. The tissue culture may becapable of regenerating plants having all the physiological andmorphological characteristics of the foregoing watermelon plant, and ofregenerating plants having substantially the same genotype as theforegoing watermelon plant. The regenerable cells in such tissuecultures may be embryos, protoplasts, meristematic cells, callus,pollen, leaves, ovules, anthers, cotyledons, hypocotyl, pistils, roots,root tips, flowers, seeds, plant, petiole, or stems. Still a furtherembodiment provides for watermelon plants regenerated from the tissuecultures of inbred watermelon line WDL0090.

Another embodiment relates to a method of vegetatively propagatinginbred watermelon line WDL0090 comprising the steps of: (a) collectingtissue capable of being propagated from the plant; (b) cultivating saidtissue to obtain proliferated shoots; and (c) rooting said proliferatedshoots to obtain rooted plantlets.

Another embodiment provides for a method for producing a hybrid seed ofhybrid line XWT8009, WT8018 or XWT8028 from inbred watermelon lineWDL0090 comprising the steps of: (a) crossing female inbred lineWTL0089, WTL0088 or WTL0091 with the male inbred line WDL0090, and (b)allowing seed of hybrid watermelon XWT8009, WT8018 or XWT8028 to form.

Another embodiment provides for a method for producing a hybrid seedfrom inbred watermelon line WDL0090 comprising the steps of: (a)crossing the watermelon plant with another inbred watermelon plant, and(b) allowing seed of a hybrid watermelon plant to form.

Another embodiment provides for a method for producing a seed of awatermelon plant derived from inbred watermelon line WDL0090 comprisingthe steps of: (a) crossing the watermelon plant with itself or a secondwatermelon plant, and (b) allowing seed of an inbred WDL0090-derivedwatermelon plant to form.

Further embodiments provide for a method of producing food or feedcomprising: (a) obtaining a plant of inbred watermelon line WDL0090,wherein the plant has been cultivated to maturity, and (b) collecting atleast one watermelon from the plant.

In another embodiment, the first step in “crossing” comprises plantingseeds of a first and second parent watermelon plant, often in proximityso that pollination will occur for example, mediated by insect vectors.Alternatively, pollen can be transferred manually. Where the plant isself-pollinated, pollination may occur without the need for direct humanintervention other than plant cultivation. A second step may comprisecultivating or growing the seeds of first and second parent watermelonplants into plants that bear flowers. A third step may comprisepreventing self-pollination of the plants, such as by emasculating theflowers (i.e., killing or removing the pollen). A fourth step for ahybrid cross may comprise cross-pollination between the first and secondparent watermelon plants. Yet another step comprises harvesting theseeds from at least one of the parent watermelon plants. The harvestedseed can be grown to produce a watermelon plant or hybrid tomato plant.

Another embodiment provides for a method for developing a watermelonplant in a plant breeding program, comprising applying plant breedingtechniques to the plant, or plant part thereof, to inbred watermelonline WDL0090, comprising crossing, recurrent selection, mutationbreeding, wherein said mutation breeding selects for a mutation that isspontaneous or artificially induced, backcrossing, pedigree breeding,marker enhanced selection, haploid/double haploid production, ortransformation to inbred watermelon line WDL0090, or its parts, whereinapplication of said techniques results in development of a watermelonplant.

Another embodiment provides for a method of introducing a mutation intothe genome of inbred watermelon line WDL0090, said method comprisingmutagenesis of the plant, or plant part thereof, of watermelon plantWDL0090, wherein said mutagenesis is selected from the group consistingof temperature, long-term seed storage, tissue culture conditions,ionizing radiation, chemical mutagens, or targeting induced locallesions in genomes, and wherein the resulting plant comprises at leastone genome mutation.

Another embodiment provides for a method of editing the genome ofwatermelon plant WDL0090, said method comprising editing the genome ofthe plant, or plant part thereof, of watermelon plant WDL0090, whereinsaid method is selected from the group comprising zinc finger nucleases,transcription activator-like effector nucleases (TALENs), engineeredhoming endonucleases/meganucleases, and the clustered regularlyinterspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9)system.

The watermelon seed of WDL0090 may be provided as an essentiallyhomogeneous population of seed of inbred watermelon line WDL0090.Essentially homogeneous populations of seed are generally free fromsubstantial numbers of other seed.

Another embodiment provides for the genetic complement of inbredwatermelon line WDL0090. The phrase “genetic complement” is used torefer to the aggregate of nucleotide sequences, the expression of whichsequences defines the phenotype of, in the present case, a watermelonplant, or a cell or tissue of that plant. A genetic complement thusrepresents the genetic makeup of a cell, tissue or plant, and a hybridgenetic complement represents the genetic make-up of a hybrid cell,tissue or plant. Thus, another embodiment provides for watermelon plantcells that have a genetic complement in accordance with the watermelonplant cells disclosed herein, and seeds and plants containing suchcells.

As used herein, “at least one,” “one or more,” and “and/or” areopen-ended expressions that are both conjunctive and disjunctive inoperation. For example, each of the expressions “at least one of A, Band C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “oneor more of A, B, or C” and “A, B, and/or C” means A alone, B alone, Calone, A and B together, A and C together, B and C together, or A, B andC together.

Various embodiments are set forth in the Detailed Description asprovided herein and as embodied by the claims. It should be understood,however, that this Summary does not contain all of the aspects andembodiments, is not meant to be limiting or restrictive in any manner,and that embodiment(s) as disclosed herein is/are understood by those ofordinary skill in the art to encompass obvious improvements andmodifications thereto.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Cotyledon. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

Embryo. The embryo is the small plant contained within a mature seed.

Gene. Gene refers to a segment of nucleic acid. A gene can be introducedinto a genome of a species, whether from a different species or from thesame species, using transformation or various breeding methods.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

Locus. Locus or loci (plural) refers to a position in the genome for agene, SNP, mutation, etc.

Plant Part(s). Plant part(s) (or a watermelon plant, or a part thereof)includes but is not limited to, regenerable cells in such tissuecultures may be embryos, taproot, protoplasts, cells, meristematiccells, callus, pollen, leaves, ovules, embryos, anthers, cotyledons,hypocotyl, pistils, roots, rootstock, root tips, scion, stock, taproots,flowers, fruit, seeds, shoot, plant, petiole, or stems.

Progeny. Progeny includes an F₁ watermelon plant produced from the crossof two watermelon plants where at least one plant includes inbredwatermelon line WDL0090 and progeny further includes, but is not limitedto, subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, and F₁₀ generationalcrosses.

Rootstock. Rootstock or stock refers to the plant selected for itsroots, in particular for the resistance of the roots to diseases orstress (e.g., heat, cold, salinity etc.). Normally the quality of thefruit of the plant providing the rootstock is less important.

Scion. Scion refers to a part of the plant attached to the rootstock.This plant is selected for its stems, leaves, flowers, or fruits. Thescion contains the desired genes to be duplicated in future productionby the stock/scion plant and may produce the desired tomato fruit.

Stock/scion. Stock/scion or grafted plant refers to a tomato plantcomprising a rootstock from one plant grafted to a scion from anotherplant.

DETAILED DESCRIPTION

Inbred watermelon lines WTL0088, WTL0089, WDL0090 and WTL0091 originatedfrom a private breeding program in the state of Florida. Inbredwatermelon line WTL0088, WTL0089, and WTL0091 are female inbredwatermelon lines and inbred watermelon line WDL0090 is a male inbredwatermelon line.

Inbred watermelon lines WTL0088, WTL0089, WDL0090 and WTL0091 have shownuniformity and stability, as described in the following varietydescription information. Inbred watermelon lines WTL0088, WTL0089,WDL0090 and WTL0091 were tested for uniformity and stability asufficient number of generations with careful attention to uniformity ofplant type and has been increased with continued observation foruniformity.

Inbred watermelon lines WTL0088, WTL0089, WDL0090 and WTL0091 have thefollowing morphologic and other characteristics based primarily on datacollected in Woodland, Calif., Aug. 16-17, 2020 as shown in Table 1.

TABLE 1 VARIETY DESCRIPTION INFORMATION Characteristic WTL0088 WTL0091WTL0089 WDL0090 Type Female inbred Female inbred Female inbred Maleinbred Ploidy Tetraploid Tetrapioid Tetraploid Diploid Maturity AverageAverage Average Average Plant vigor Excellent Excellent Excellent Verygood Leaf type Lobed Lobed Lobed Non-lobed Sex expression MoneciousMonoecious Monoecious Monoecious Stem Stem shape (cross section)Circular Circular Circular Circular Diameter at second node 6.0 mm 7.0mm 7.0 mm 6.0 mm Stem surface Pubescent Pubescent Pubescent PubescentVine length at harvest 225 cm 215 cm 228 cm 210 cm Number of internodesat harvest 34 26 29 22 Ratio of vine length to internodes 6.62 8.27 7.869.55 Leaf Leaf shape Obovate Obovate Obovate Obovate Leaf lobes LobedLobed Lobed Lobed Leaf length 15.0 cm 12.3 cm 15.8 cm 13.4 cm Leaf width18.4 cm 14.4 cm 16.1 cm 9.8 cm Leaf size (length:width) 0.82 0.85 0.981.37 Dorsal surface pubescence Pubescent Slightly pubescent PubescentVery Slight Pubescence Ventral surface pubescence Pubescent PubescentPubescent Very Slight Pubescence Leaf color (upper) RHS 147B RHS NN137BRHS NN137B RHS NN137B Leaf color (lower) Closest to RHS 147C Closest tobut lighter Closest to but lighter Closest to RHS 147B than RHS 147Bthan RHS 147B Flower Diameter of staminate 3.1 cm 2.9 cm 3.3 cm 3.3 cmDiameter of pistilate 3.0 cm 2.8 cm 3.1 cm 3.0 cm Flower color RHS 5BRHS 5A RHS 5A RHS 6A Fruit Fruit shape Round Round Round Oval Length25.0 cm 26.8 cm 27.4 46 Diameter at midsection 26.8 cm 29.0 cm 26 22.8Average weight 14.45 lbs 23.6 lbs 20.67 lbs 28 lbs Fruit surface SmoothSmooth Smooth Smooth Rind pattern Crimson sweet Crimson sweet CrimsonSweet All sweet Rind background color Medium dark green Medium darkgreen Medium dark green Light green Rind primary color Closest to butdarker Closest to but darker Closest to but darker 139A than RHS 139Athen RHS 139A than 139A Rind stripe color Dark green Dark green DarkGreen Medium Dark Green Rind secondary color RHS 143A RHS 143A 143A 144ARind tertiary color 145B 145C 145C 145B Rind stripe width Medium MediumMedium Wide Rind texture Tough Tough Tough Tough Rind thickness atblossom end 21.0 mm 20.0 mm 15.0 mm 8.0 mm Rind thickness - sides 17.0mm 15.0 mm 12.0 mm 13.0 mm Internal/Flesh Flesh texture Crisp CrispCrisp Crisp Flesh coarseness Fine-little fiber Fine-little fiberFine-little fiber Fine-little fiber Flesh color RHS 35A with RHS 42B RHS35A with RHS 42B RHS 42B RHS 42B Flesh color Deep red Deep red Deep RedBright Red Seed Seed color Tipped, closest to Mottled Black; Striped;RHS 203A edges RHS 165B with RHS 200A and RHS 164A with with RHS 164ARHS 200A at tip RHS 165A RHS 200B in the middle Seed size Medium MediumMedium Small Seed length 10.0 mm 10.0 mm 11.0 mm 8.0 mm Seed width 7.5mm 7.5 mm 7.0 mm 5.0 mm Seed thickness 2.5 mm 3.0 mm 2.5 mm 2.0 mm Seedyield per fruit 56 200 140 588 Disease resistance Disease resistanceFusarium oxysporum Fusarium oxysporum Fusarium oxysporum Fusariumoxysporum f. sp. niveum (FON) f. sp. niveum (FON) f. sp. niveum (FON) f.sp. niveum (FON) race 1 race 1 race 1 race 1 and Colletotrichumorbiculare

Table 2 below provides a comparison of penetrometer or flesh firmness ofinbred watermelon lines WTL0088, WTL0089, WDL0090 and WTL0091. Apenetrometer is a device that is used to determine the firmness of theflesh of watermelon by punching holes in the flesh of the watermelon. Astandard penetrometer with an 11 mm wide probe was used. Ten fruit werecut in half length wise and a penetrometer was used to puncture theflesh in a cross shape on the center of each watermelon half in the samelocation and the same speed with ten readings on each inbred. Table 2below shows the results of the ten reading on each inbred as well as thestatistical analysis of the data.

TABLE 2 Watermelon Firmness Measured by a Penetrometer WTL0088 WTL0091WTL0089 WDL0090 1 3.4 5.1 4.4 3.6 2 2.8 4 3.5 4 3 4.5 4 2.8 4.3 4 5.5 54.1 3.3 5 4.6 4.3 2.7 2.2 6 2.3 2.8 2.7 2.2 7 3.3 3.1 2.6 2.8 8 2.4 2.53 2.4 9 2.7 3.8 3.5 3 10 2.2 3.8 4.4 5 Mean 3.18 3.84 3.37 3.28 Standarderror 0.19 0.27 0.23 0.30 Median 3.20 3.90 3.25 3.15 Mode 3.20 4.00 4.402.20 Standard deviation 0.61 0.86 0.72 0.94 Sample variance 0.37 0.740.52 0.89

Inbred watermelon lines WTL0088, WTL0089, WDL0090 and WTL0091 as well asthe hybrid lines XWT8009, WT8018, and XWT8028 described below may alsobe grown for use as rootstocks (stocks) or scions. Typically, differenttypes of watermelon are grafted to enhance disease resistance, which isusually conferred by the rootstock, while retaining the horticulturalqualities usually conferred by the scion. It is not uncommon forgrafting to occur between cultivated watermelon varieties and relatedwatermelon species. Methods of grafting and vegetative propagation arewell-known in the art.

In another aspect, the disclosure provides to a plant comprising arootstock or scion of WTL0088, WTL0089, WDL0090 and WTL0091 as well asthe hybrid lines XWT8009, WT8018, and XWT8028.

One aspect of the current disclosure concerns methods for hybridwatermelon seed produced from breeding with inbred line WTL0088 as aparental line. In one example, inbred line WTL0088 is used as a femaleline and crossed with the male inbred watermelon WDL0090 to produce thehybrid XWT8009 (described below in Table 3). Alternatively, in otherembodiments, inbred line WTL0088 may be crossed with another inbred lineto produce additional hybrid lines. Additionally, inbred line WTL0088,hybrid line XWT8009 or inbred line WDL0090 may be may be crossed withitself or with any second plant. Such methods can be used forpropagation of hybrid XWT8009, and/or the watermelon lines WTL0088 andWDL0090, or can be used to produce plants that are derived from hybridXWT8009, and/or the watermelon lines WTL0088 and WDL0090. Plants derivedfrom hybrid XWT8009, and/or the watermelon lines WTL0088 and WDL0090 maybe used, in certain embodiments, for the development of new watermelonvarieties.

Another aspect of the current disclosure concerns methods for hybridwatermelon seed produced from breeding with inbred line WTL0089 as aparental line. In one example, inbred line WTL0089 is used as a femaleline and crossed with the male inbred watermelon WDL0090 to produce thehybrid XWT8028 (described below in Table 3). Alternatively, in otherembodiments, inbred line WTL0089 may be crossed with another inbred lineto produce additional hybrid lines. Additionally, inbred line WTL0089,hybrid line XWT8028 or inbred line WDL0090 may be may be crossed withitself or with any second plant. Such methods can be used forpropagation of hybrid XWT8028, and/or the watermelon lines WTL0089 andWDL0090, or can be used to produce plants that are derived from hybridXWT8028, and/or the watermelon lines WTL0089 and WDL0090. Plants derivedfrom hybrid XWT8028, and/or the watermelon lines WTL0089 and WDL0090 maybe used, in certain embodiments, for the development of new watermelonvarieties.

One aspect of the current disclosure concerns methods for hybridwatermelon seed produced from breeding with inbred line WTL0091 as aparental line. In one example, inbred line WTL0091 is used as a femaleline and crossed with the male inbred watermelon WDL0090 to produce thehybrid WT8018 (described below in Table 3). Alternatively, in otherembodiments, inbred line WTL0091 may be crossed with another inbred lineto produce additional hybrid lines. Additionally, inbred line WTL0091,hybrid line WT8018 or inbred line WDL0090 may be may be crossed withitself or with any second plant. Such methods can be used forpropagation of hybrid XWT8018, and/or the watermelon lines WTL0091 andWDL0090, or can be used to produce plants that are derived from hybridWT8018, and/or the watermelon lines WTL0091 and WDL0090. Plants derivedfrom hybrid WT8018, and/or the watermelon lines WTL0091 and WDL0090 maybe used, in certain embodiments, for the development of new watermelonvarieties.

Hybrid watermelon lines XWT8009, WT8018, and XWT8028, as describedabove, originated from a private breeding program in the state ofFlorida. Hybrid watermelon lines XWT8009, WT8018, and XWT8028 have thefollowing morphologic and other characteristics based primarily on datacollected in Woodland, Calif. 2020 as shown in Table 3.

Table 4 provide a comparison of hybrid watermelon lines XWT8009, WT8018,and XWT8028 with the commercial hybrid triploid watermelon linesCharismatic, Troubador, Traveler, Fascination, Unbridled, Exclamation,Maxima, and Captivation. The comparison trial was conducted in Woodland,Calif. in 2019.

TABLE 3 HYBRID DESCRIPTION INFORMATION Characteristic XWT8009 XWT8018XWT8028 Mature Fruit Fruit Shape Oval Oval Oval cm long 30.7 cm 32.5 cm32.5 cm cm diameter at midsection 26.6 cm 26.8 cm 24.6 cm lbs avg.weight 17.93 lbs 20.33 lbs 22.87 lbs Index (length/diameter)*10) 11.5412.13 13.21 Fruit Surface Smooth Smooth Smooth Skin Color Pattern StripeStripe Stripe Primary color Closest to but darker Closest to but darkerClosest to but darker than 139A than 139A than 139A (Dark YellowishGreen) (Dark Yellowish Green) Secondary Color 143A 144A Closest to butdarker (Strong Yellow Green) than 143A (Strong Yellow Green) TertiaryColor 145C Closest to 145B Closest to 145B Rind Rind Texture Tough ToughTough Thickness at blossom end 12 mm 16 mm 10 mm Thickness sides 11 mm11 mm 14.5 mm 18 mm on one side, 11 mm on other side Flesh Flesh TextureCrisp Crisp Crisp Flesh Coarseness Fine-Little Fiber Fine-Little FiberFine-Little Fiber Flesh Color 42A with 39A 42B with 42C Between 42A and42B Disease Resistance Fo Race 1 Fo Race 1 Fo Race 1 Penetrometer 2.934.73 3.00

TABLE 4 COMPARISON OF HYBRIDS WITH COMMERICAL TRIPLOID LINESCharacteristic XWT8009 XWT8018 XWT8028 Charismatic Troubador TravelerGeneral Fruit Type Oblong Oblong Oblong Oblong Oblong Oblong PloidyTriploid Triploid Triploid Triploid Triploid Triploid Stem Stem Shape(in Circular Circular Circular Circular Circular Circular Cross section)mm diameter at 5 7 7 6 5 7 second node Stem Surface Bristled PubescentPubescent Pubescent Pubescent Pubescent cm vine length 330 345 277 285137 148 at last harvest # of internodes 36 35 31 34 11 26 at lastharvest Ratio - Vine length: 9.17 9.86 8.94 8.38 12.45 5.69 # OfInternodes Leaf cm leaf length 13.47 12.4 13.73 14.23 12.77 15.7 cm leafwidth 12.87 11.8 13.73 11.37 10.57 13.43 Leaf size (length:Width) 1.051.05 1.00 1.25 1.21 1.17 Leaf color Upper: RHS 147B RHS NN137C Upper:RHS 147A RHS N148A RHS 147A (Moderate (Greyish NN137B (Moderate(Moderate (Moderate Yellow Green) Olive Green) (Greyish Olive Green)Yellow Green) Olive Green) Olive Green) Lower: Lower: RHS 147C 191A(Moderate (Greyish Yellow Green) Yellow Green) Flower cm diameterStaminate 3.5 2.8 2.7 3.6 4.1 3.3 cm diameter Pistilate 3.6 3.8 2.5 3.83.8 2.5 Flower Color RHS 8A RHS 9B RHS 6B RHS 4B RHS 3C RHS 4B(Brilliant (Vivid (Brilliant (Light (Light (Light Yellow) Yellow)Greenish Greenish Greenish Greenish Yellow) Yellow) Yellow) Yellow)Mature Fruit Fruit Shape Oval Oval Oval Oval Oval Oval cm long 27 30 2827 23 24 cm diameter at 22 22 23 25 20 22 midsection lbs avg. weight18-20 22-24 20-22 16-20 14-17 15-20 Index (length/ 12.27 13.64 12.1710.80 11.50 10.91 diameter) *10) Primary color Closest to but Lighterthan Closest to but Between RHS 139A Closest to but darker than RHS 139Amuch darker RHS 146B and lighter than RHS 139A than RHS 139A RHS 146ARHS 139A (Dark Yellowish Green) Secondary Color RHS 143A RHS 144C RHS143A RHS N144C RHS 144A RHS 138A (Strong (Strong (Strong (Strong YellowGreen) Yellow Green) Yellow Green) Yellow Green) Tertiary Color RHS 145CRHS 145D RHS 145C RHS 194C RHS 144D RHS 147D Rind Rind Texture ToughTough Tough Tough Tough Tough mm thickness at 22 17 16 17 25 35 blossomend mm thickness sides 14 11 12 12 17 15 Flesh Flesh Texture Crisp CrispCrisp Crisp Crisp Crisp Flesh Coarseness Fine-Little Fine-LittleFine-Little Fine-Little Fine-Little Fine-Little Fiber Fiber Fiber FiberFiber Fiber Flesh Color 44A Closest to but 44C Between 42A 45B lighterthan 42C and 42D 42A Disease Resistance Fo Race 1 Fo Race 1 Fo Race 1HR: Co (1) Penetrometer 4.05 3.68 3.78 3.53 4.18 3.43 CharacteristicFascination Unbridled Exclamation Maxima Captivation General Fruit TypeOblong Oblong Oblong Oblong Oblong Ploidy Triploid Triploid TriploidTriploid Triploid Stem Stem Shape (in Circular Circular CircularCircular Circular Cross section) mm diameter at 7 5 7 7 8 second nodeStem Surface Bristled Pubescent Pubescent Bristled Bristled cm vinelength at 160 92 150 220 140 last harvest # of internodes at 22 16 15 2218 last harvest Ratio - Vine length: 7.27 5.75 10.00 10.00 7.78 # OfInternodes Leaf cm leaf length 16.87 15.53 13.33 12.07 14.7 cm leafwidth 16.33 14.33 13.5 11.8 12.8 Leaf size (length:Width) 1.03 1.08 0.991.02 1.15 Leaf color RHS N148A RHS NN137B RHS 137C RHS NN137A RHS 147A(Moderate (Greyish (Moderate (Greyish (Moderate Yellow Green) OliveGreen) Yellow Green) Olive Green) Olive Green) Flower cm diameterStaminate 2 3.9 2.5 3 4 cm diameter Pistilate 2 3.6 2.8 3 3.5 FlowerColor RHS 8B RHS 5B RHS 6B RHS 3A RHS 4A (Light (Brilliant (Brilliant(Brilliant (Brilliant Greenish Greenish Greenish Greenish GreenishYellow) Yellow) Yellow) Yellow) Yellow) Mature Fruit Fruit Shape OvalCylindrica1 Cylindrical Cylindrical Cylindrical cm long 28 25 25 26 25cm diameter at 23 22 22 24 21 midsection lbs avg. weight 16-20 18-2217-21 19-22 14-17 Index (length/ 12.17 11.36 11.36 10.83 11.90 diameter)*10) Primary color RHS 137A RHS 137B RHS 137B RHS 139A RHS 137A(Moderate Olive Green) Secondary Color RHS 145A RHS 144A RHS 144C RHS143A RHS 143C (Strong (Strong Yellow Green) Yellow Green) Tertiary ColorRHS 149D RHS 147D RHS 145D RHS 145B RHS 145D (Light Yellow Green) RindRind Texture Tough Tough Tough Tough Tough mm thickness at 16 28 16 3017 blossom end mm thickness sides 14 15 12 15 15 Flesh Flesh TextureCrisp Crisp Crisp Crisp Crisp Flesh Coarseness Fine-Little Fine-LittleFine-Little Fine-Little Fine-Little Fiber Fiber Fiber Fiber Fiber FleshColor Slightly darker Closest to 42A Closest to but 42B than 42D RHS 42Clighter than (Strong 42A Reddish Orange) Disease Resistance IR: Co (1)IR: Co (1) IR: Co (1) IR: Fo (1) IR: Fo (1) IR: Fo (1) Penetrometer 3.433.00 3.82 3.75 3.85Breeding With Inbred Watermelon Line WTL0088, WTL0089, WDL0090 orWTL0091

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable variety. This approach hasbeen used extensively for breeding disease-resistant varieties. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial varieties; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardvariety. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of watermelon breeding is to develop new and superiorwatermelon varieties and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selection, selfing and mutations.

Using Inbred Watermelon Lines WTL0088, WTL0089, WDL0090 and WTL0091 toDevelop Other Watermelon Varieties

Watermelon varieties such as inbred watermelon lines WTL0088, WTL0089,WDL0090 or WTL0091 are typically developed for the use as parental linesin the production of triploid hybrids for fresh market consumption.Plant breeding techniques known in the art and used in a watermelonbreeding program include, but are not limited to, recurrent selection,mass selection, bulk selection, mass selection, backcrossing, pedigreebreeding, open pollination breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection,making double haploids, transformation, and gene editing. Thesetechniques can be used singularly or in combinations. The development ofwatermelon varieties in a breeding program requires, in general, thedevelopment and evaluation of homozygous varieties. There are manyanalytical methods available to evaluate a new variety. The oldest andmost traditional method of analysis is the observation of phenotypictraits, but genotypic analysis may also be used.

Additional Breeding Methods

One embodiment is directed to methods for producing a watermelon plantby crossing a first parent watermelon plant with a second parentwatermelon plant, wherein the first or second watermelon plant is thewatermelon plant from inbred watermelon lines WTL0088, WTL0089, WDL0090or WTL0091. Further, both first and second parent watermelon plants maybe from inbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091. Anyplants produced using inbred watermelon line WTL0088, WTL0089, WDL0090or WTL0091 as at least one parent are also within the scope of theembodiments. These methods are well known in the art and some of themore commonly used breeding methods are described herein. Descriptionsof breeding methods can be found in one of several reference books(e.g., Allard, Principles of Plant Breeding (1960); Simmonds, Principlesof Crop Improvement (1979); Sneep, et al. (1979); Cooper, S. G., D. S.Douches and E. J. Grafius. 2004.

The following describes breeding methods that may be used with inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091 in the developmentof further watermelon plants, including inbreds or hybrids. One suchembodiment is a method for developing an inbred watermelon line WTL0088,WTL0089, WDL0090 or WTL0091 progeny plant in a watermelon breedingprogram comprising: obtaining the watermelon plant, or a part thereof,of inbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091,utilizing said plant, or plant part, as a source of breeding material,and selecting an inbred watermelon line WTL0088, WTL0089, WDL0090 orWTL0091 progeny plant with molecular markers in common with inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091 and/or withmorphological and/or physiological characteristics selected from thecharacteristics listed in Table 1. Breeding steps that may be used inthe watermelon plant breeding program include pedigree breeding,backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example, SSR markers), and the making ofdouble haploids may be utilized.

Another method involves producing a population of inbred watermelon lineWTL0088, WTL0089, WDL0090 or WTL0091 progeny watermelon plants,comprising crossing inbred watermelon line WTL0088, WTL0089, WDL0090 orWTL0091 with another watermelon plant, thereby producing a population ofwatermelon plants which derive 50% of their alleles from inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091. A plant of thispopulation may be selected and repeatedly selfed or sibbed with aninbred watermelon line resulting from these successive filialgenerations. One embodiment is the inbred watermelon line produced bythis method and that has obtained at least 50% of its alleles frominbred watermelon lines WTL0088, WTL0089, WDL0090 or WTL0091.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see, Fehr and Walt, Principles of VarietyDevelopment, pp. 261-286 (1987). Thus, embodiments include inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091 progeny watermelonplants comprising a combination of at least two inbred watermelon lineWTL0088, WTL0089, WDL0090 or WTL0091 traits selected from the groupconsisting of those listed in Table 1 and a combination of traits listedin the Summary, so that said progeny watermelon plant is notsignificantly different for said traits than inbred watermelon lineWTL0088, WTL0089, WDL0090 or WTL0091 as determined at the 5%significance level when grown in the same environmental conditions.Using techniques described herein, molecular markers may be used toidentify said progeny plant as an inbred watermelon line WTL0088,WTL0089, WDL0090 or WTL0091 progeny plant. Mean trait values may be usedto determine whether trait differences are significant, and preferablythe traits are measured on plants grown under the same environmentalconditions. Once such a variety is developed, its value is substantialsince it is important to advance the germplasm base as a whole in orderto maintain or improve traits such as yield, disease tolerance, pesttolerance, and plant performance in extreme environmental conditions.

Progeny of inbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091may also be characterized through their filial relationship with inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091, as for example,being within a certain number of breeding crosses of inbred watermelonline WTL0088. A breeding cross is a cross made to introduce new geneticsinto the progeny, and is distinguished from a self or a sib cross, whichis made to select among existing genetic alleles. The lower the numberof breeding crosses in the pedigree, the closer the relationship betweeninbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091 and itsprogeny. For example, progeny produced by the methods described hereinmay be within 1, 2, 3, 4, or 5 breeding crosses of inbred watermelonline WTL0088, WTL0089, WDL0090 or WTL0091.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asinbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091 and anotherinbred watermelon line having one or more desirable characteristics thatis lacking or which complements inbred watermelon line WTL0088, WTL0089,WDL0090 or WTL0091. If the two original parents do not provide all thedesired characteristics, other sources can be included in the breedingpopulation. In the pedigree method, superior plants are selfed andselected in successive filial generations. In the succeeding filialgenerations, the heterozygous condition gives way to homogeneousvarieties as a result of self-pollination and selection. Typically, inthe pedigree method of breeding, five or more successive filialgenerations of selfing and selection is practiced: F₁ to F₂; F₂ to F₃;F₃ to F₄; F₄ to F₅; etc. After a sufficient amount of inbreeding,successive filial generations will serve to increase seed of thedeveloped variety. Preferably, the developed variety compriseshomozygous alleles at about 95% or more of its loci.

Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous variety orinbred line which is the recurrent parent. The source of the trait to betransferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., variety) and the desirable trait transferred from thedonor parent. This is also known as single gene conversion and/orbackcross conversion.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, when compared in the same environment, other than an occasionalvariant trait that might arise during backcrossing or directintroduction of a transgene, and therefore the desired physiological andmorphological constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance, itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

A backcross conversion of inbred watermelon line WTL0088, WTL0089,WDL0090 or WTL0091 occurs when DNA sequences are introduced throughbackcrossing, with inbred watermelon line WTL0088 utilized as therecurrent parent. Both naturally occurring and transgenic DNA sequencesmay be introduced through backcrossing techniques. A backcrossconversion may produce a plant with a trait or locus conversion in atleast two or more backcrosses, including at least 2 crosses, at least 3crosses, at least 4 crosses, at least 5 crosses, and the like. Molecularmarker assisted breeding or selection may be utilized to reduce thenumber of backcrosses necessary to achieve the backcross conversion. Forexample, see, Frisch M. et al, “Marker-Assisted Backcrossing forSimultaneous Introgression of Two Genes” Crop Science Society ofAmerica, pp 1716-1725 (2001) and Openshaw, S. J., et al.,“Marker-assisted Selection in Backcross Breeding, Proceedings Symposiumof the Analysis of Molecular Data” Crop Science Society of America,Corvallis, Oreg. (August 1994), where it was demonstrated that abackcross conversion could be made in as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes ascompared to unlinked genes), the level of expression of the trait, thetype of inheritance (cytoplasmic or nuclear), and the types of parentsincluded in the cross. It is understood by those of ordinary skill inthe art that for single gene traits that are relatively easy toclassify, the backcross method is effective and relatively easy tomanage. Desired traits that may be transferred through backcrossconversion include, but are not limited to, sterility (nuclear andcytoplasmic), fertility restoration, nutritional enhancements, droughttolerance, nitrogen utilization, low phytate, industrial enhancements,disease tolerance (bacterial, fungal, or viral), insect tolerance, andherbicide tolerance. In addition, an introgression site itself, such asan FRT site, Lox site, or other site-specific integration site, may beinserted by backcrossing and utilized for direct insertion of one ormore genes of interest into a specific plant variety. In someembodiments, the number of loci that may be backcrossed into inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091 is at least 1, 2,3, 4, or 5, and/or no more than 6, 5, 4, 3, or 2. A single locus maycontain several transgenes, such as a transgene for disease tolerancethat, in the same expression vector, also contains a transgene forherbicide tolerance. The gene for herbicide tolerance may be used as aselectable marker and/or as a phenotypic trait. A single locusconversion of site-specific integration system allows for theintegration of multiple genes at the converted loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, “Breeding Field Crops”p. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant, and easily recognized traits.

One process for adding or modifying a trait or locus in inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091 comprises crossinginbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091 plants grownfrom inbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091 seedwith plants of another inbred watermelon line that comprise the desiredtrait or locus, selecting F₁ progeny plants that comprise the desiredtrait or locus to produce selected F₁ progeny plants, crossing theselected progeny plants with the inbred watermelon line WTL0088,WTL0089, WDL0090 or WTL0091 plants to produce backcross progeny plants,selecting for backcross progeny plants that have the desired trait orlocus and the morphological characteristics of inbred watermelon lineWTL0088, WTL0089, WDL0090 or WTL0091 to produce selected backcrossprogeny plants, and backcrossing to inbred watermelon line WTL0088,WTL0089, WDL0090 or WTL0091 three or more times in succession to produceselected fourth or higher backcross progeny plants that comprise saidtrait or locus. The modified inbred watermelon line WTL0088, WTL0089,WDL0090 or WTL0091 may be further characterized as having thephysiological and morphological characteristics of inbred watermelonline WTL0088, WTL0089, WDL0090 or WTL0091 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or may be characterized by percentsimilarity or identity to inbred watermelon line WTL0088, WTL0089,WDL0090 or WTL0091 as determined by SSR markers. The above method may beutilized with fewer backcrosses in appropriate situations, such as whenthe donor parent is highly related or markers are used in the selectionstep. Desired traits that may be used include those nucleic acids knownin the art, some of which are listed herein, that will affect traitsthrough nucleic acid expression or inhibition. Desired loci include theintrogression of FRT, Lox, and other sites for site specificintegration, which may also affect a desired trait if a functionalnucleic acid is inserted at the integration site.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny watermelon seedby adding a step at the end of the process that comprises crossinginbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091 with theintrogressed trait or locus with a different watermelon plant andharvesting the resultant first generation progeny watermelon seed.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques well-known in the art. Single genetraits may or may not be transgenic. Examples of these traits include,but are not limited to, herbicide tolerance, insect tolerance, tolerancefor bacterial, fungal, or viral disease, male fertility, male sterility,enhanced nutritional quality, modified carbohydrate metabolism, modifiedyield, modified glycoalkaloid content, and industrial usage

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodagronomic characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent, but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. For example, aninbred watermelon line may be crossed with another variety to produce afirst-generation progeny plant. The first-generation progeny plant maythen be backcrossed to one of its parent varieties to create a BC₁ orBC₂. Progeny are selfed and selected so that the newly developed varietyhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the nonrecurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newwatermelon varieties.

Therefore, an embodiment of the present disclosure is a method of makinga backcross conversion inbred watermelon line WTL0088, WTL0089, WDL0090or WTL0091, comprising the steps of crossing a plant of inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091 with a donor plantcomprising a desired trait, selecting an F₁ progeny plant comprising thedesired trait, and backcrossing the selected F₁ progeny plant to a plantof inbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091 toproduce BC₁, BC₂, BC₃, etc. This method may further comprise the step ofobtaining a molecular marker profile of inbred watermelon line WTL0088,WTL0089, WDL0090 or WTL0091 and using the molecular marker profile toselect for a progeny plant with the desired trait and the molecularmarker profile of inbred watermelon line WTL0088, WTL0089, WDL0090 orWTL0091. In one embodiment, the desired trait is a mutant gene, gene, ortransgene present in the donor parent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Inbred watermelon line WTL0088, WTL0089,WDL0090 or WTL0091 is suitable for use in a recurrent selection program.The method entails individual plants cross pollinating with each otherto form progeny. The progeny are grown and the superior progeny selectedby any number of selection methods, which include individual plant,half-sib progeny, full-sib progeny, and selfed progeny. The selectedprogeny are cross pollinated with each other to form progeny for anotherpopulation. This population is planted and again superior plants areselected to cross pollinate with each other. Recurrent selection is acyclical process and therefore can be repeated as many times as desired.The objective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain new varieties for commercial or breedinguse, including the production of a synthetic variety. A syntheticvariety is the resultant progeny formed by the intercrossing of severalselected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self-pollination, directed pollinationcould be used as part of the breeding program.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Mutation Breeding

Mutation breeding is another method of introducing new traits intoinbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091. Mutationsthat occur spontaneously or are artificially induced can be usefulsources of variability for a plant breeder. The goal of artificialmutagenesis is to increase the rate of mutation for a desiredcharacteristic. Mutation rates can be increased by many different meansincluding temperature, long-term seed storage, tissue cultureconditions, radiation; such as X-rays, Gamma rays (e.g., cobalt 60 orcesium 137), neutrons, (product of nuclear fission by uranium 235 in anatomic reactor), Beta radiation (emitted from radioisotopes such asphosphorus 32 or carbon 14), or ultraviolet radiation (preferably from2500 to 2900 nm), or chemical mutagens (such as base analogues(5-bromo-uracil)), related compounds (8-ethoxy caffeine), antibiotics(streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards,epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones),azide, hydroxylamine, nitrous acid, or acridines. Once a desired traitis observed through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Fehr, “Principles of VarietyDevelopment,” Macmillan Publishing Company (1993). In addition,mutations created in other watermelon plants may be used to produce abackcross conversion of inbred watermelon line WTL0088, WTL0089, WDL0090or WTL0091 that comprises such mutation.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the embodiments areintended to be within the scope of the embodiments.

Gene Editing Using CRISPR

Targeted gene editing can be done using CRISPR/Cas9 technology (Saunders& Joung, Nature Biotechnology, 32, 347-355, 2014). CRISPR is a type ofgenome editing system that stands for Clustered Regularly InterspacedShort Palindromic Repeats. This system and CRISPR-associated (Cas) genesenable organisms, such as select bacteria and archaea, to respond to andeliminate invading genetic material. Ishino, Y., et al. J. Bacteriol.169, 5429-5433 (1987). These repeats were known as early as the 1980s inE. coli, but Barrangou and colleagues demonstrated that S. thermophiluscan acquire resistance against a bacteriophage by integrating a fragmentof a genome of an infectious virus into its CRISPR locus. Barrangou, R.,et al. Science 315, 1709-1712 (2007). Many plants have already beenmodified using the CRISPR system, including Capsicum annuum. See forexample, Yildirum, Dr. Kubilay, Ondokuz Mayis University, Department ofMolecular Biology, “Genetic Conferring Multiple Resistance to DNAViruses in Plants with CRISPR/Cas9 Genome Editing Technology”, EuropeanCooperation in Science and Technology (2020), U.S. ApplicationPublication No. WO2014068346 (György et al., Identification of aXanthomonas euvesicatoria resistance gene from pepper (Capsicum annuum)and method for generating plants with resistance), Martinelli, F. etal., “Proposal of a Genome Editing System for Genetic Resistance toTomato Spotted Wilt Virus” American Journal of Applied Sciences 2014,and Noman, A. et al., “CRISPR-Cas9: Tool for Qualitative andQuantitative Plant Genome Editing” Frontiers in Plant Science Vol. 7Nov. 2016.

Gene editing can also be done using crRNA-guided surveillance systemsfor gene editing. Additional information about crRNA-guided surveillancecomplex systems for gene editing can be found in the followingdocuments, which are incorporated by reference in their entirety: U.S.Application Publication No. 2010/0076057 (Sontheimer et al., Target DNAInterference with crRNA); U.S. Application Publication No. 2014/0179006(Feng, CRISPR-CAS Component Systems, Methods, and Compositions forSequence Manipulation); U.S. Application Publication No. 2014/0294773(Brouns et al., Modified Cascade Ribonucleoproteins and Uses Thereof);Sorek et al., Annu. Rev. Biochem. 82:273-266, 2013; and Wang, S. et al.,Plant Cell Rep (2015) 34: 1473-1476.

Therefore, it is another embodiment to use the CRISPR system on inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091 to modify traitsand resistances or tolerances to pests, herbicides, and viruses.

Introduction of a New Trait or Locus into Inbred Watermelon LineWTL0088, WTL0089, WDL0090 or WTL0091

Inbred watermelon line WTL0088, WTL0089, WDL0090 or WTL0091 represents anew variety into which a new locus or trait may be introgressed. Directtransformation and backcrossing represent two important methods that canbe used to accomplish such an introgression. The term backcrossconversion and single locus conversion are used interchangeably todesignate the product of a backcrossing program.

Molecular Techniques Using Inbred Watermelon Line WTL0088, WTL0089,WDL0090 or WTL0091

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to “alter” (the utilization of up-regulation,down-regulation, or gene silencing) the traits of a plant in a specificmanner. Any DNA sequences, whether from a different species or from thesame species, which are introduced into the genome using transformationor various breeding methods are referred to herein collectively as“transgenes.” In some embodiments, a transgenic variant of inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091 may contain atleast one transgene. Over the last fifteen to twenty years severalmethods for producing transgenic plants have been developed, and anotherembodiment also relates to transgenic variants of the claimed inbredwatermelon line WTL0088, WTL0089, WDL0090 or WTL0091.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least about 1000 nucleotides of sequence upstream from the 5′end of the coding region and at least about 200 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. Less commonbases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthineand others can also be used for antisense, dsRNA and ribozyme pairing.For example, polynucleotides that contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity andto be potent antisense inhibitors of gene expression. Othermodifications, such as modification to the phosphodiester backbone, orthe 2′-hydroxy in the ribose sugar group of the RNA can also be made.The antisense polynucleotides and ribozymes can consist entirely ofribonucleotides, or can contain mixed ribonucleotides anddeoxyribonucleotides. The polynucleotides of the embodiments may beproduced by any means, including genomic preparations, cDNApreparations, in-vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

One embodiment is a process for producing inbred watermelon lineWTL0088, WTL0089, WDL0090 or WTL0091 further comprising a desired trait,said process comprising introducing a transgene that confers a desiredtrait to a watermelon plant of inbred watermelon line WTL0088, WTL0089,WDL0090 or WTL0091. Another embodiment is the product produced by thisprocess. In one embodiment, the desired trait may be one or more ofherbicide tolerance, insect tolerance, disease tolerance, or modifiedcarbohydrate metabolism. The specific gene may be any known in the artor listed herein, including: a polynucleotide conferring resistance toimidazolinone, dicamba, sulfonylurea, glyphosate, glufosinate, triazine,PPO-inhibitor herbicides, benzonitrile, cyclohexanedione, phenoxyproprionic acid, and L-phosphinothricin; a polynucleotide encoding aBacillus thuringiensis polypeptide; or a polynucleotide conferringresistance to Tomato spotted wilt virus, Xanthomonas euvesicatoria, Bs2,CARAV1, or CaPMEI1.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Li D. et al, “Establishment of a highly efficienttransformation system for pepper (Capsicum annuum L.)” Plant CellReports, pp 785-788 (2003), and Lee Y. H. et al, “A new selection methodfor pepper transformation: callus-mediated shoot formation” Plant CellReports pp 50-58 (2004). In addition, expression vectors and in vitroculture methods for plant cell or tissue transformation and regenerationof plants are available. See, for example, Gruber, et al., “Vectors forPlant Transformation,” in Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., CRC Press, Inc., Boca Raton, pp.89-119 (1993) and Nakagawa T. et al, “Development of series of gatewaybinary vectors, pGWBs, for realizing efficient construction of fusiongenes for plant transformation” Journal of Bioscience and Bioengineeringpp 34-41 (2007).

A genetic trait which has been engineered into the genome of aparticular watermelon plant may then be moved into the genome of anothervariety using traditional breeding techniques that are well known in theplant breeding arts. For example, a backcrossing approach is commonlyused to move a transgene from a transformed inbred watermelon line intoan already developed inbred watermelon line, and the resulting backcrossconversion plant would then comprise the transgene(s).

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to, genes,coding sequences, inducible, constitutive and tissue specific promoters,enhancing sequences, and signal and targeting sequences. For example,see the traits, genes, and transformation methods listed in U.S. Pat.No. 6,118,055.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Amplified Fragment Length Polymorphisms (AFLPs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), Simple SequenceRepeats (SSRs), and Single Nucleotide Polymorphisms (SNPs) may be usedin plant breeding methods utilizing inbred watermelon line WTL0088,WTL0089, WDL0090 or WTL0091.

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. See Richards, Christopher M., et al., “Polymorphicmicrosatellite markers for inferring diversity in wild and domesticatedsugar beet (Beta vulgaris), Molecular Ecology Notes (2004) 4: Pp.243-245 and Yudina, Rimma Sergeevna, “Malate dehydrogenase isozymes asmarkers of the organelles physiological state in sugar beet (Betavulgaris L.)”, March 2007, Sugar Tech, 9(1):67-71.

Randomly Amplified Polymorphic DNAs (RAPDs) and Amplified FragmentLength Polymorphisms (AFLPs) may also be used. See for example,Izzatullayeva, Vusala, “Efficiency of using RAPD and ISSR markers inevaluation of genetic diversity in sugar watermelon”, Turk. J. Biol.

(2014) 38:429-438 and Grimmer, M. K., et al., “An anchored linkage mapfor sugar watermelon based on AFLP, SNP and RAPD markers and QTL mappingof a new source of resistance to Watermelon necrotic yellow vein virus”Theor Appl Genet. (2007 May) 114(7):1151-60.

SSR technology can be routinely used. See for example, Tas̆ki-Ajduković,Ksenija, et al., “Estimation of genetic diversity and relationship insugar watermelon pollinators based on SSR markers” J. of Biotechnology(May 2017) 27: pages: 1-7.

Single Nucleotide Polymorphisms (SNPs) may also be used to identify theunique genetic composition of the embodiment(s) and progeny varietiesretaining that unique genetic composition. See for example, Stevanto,Piergiorgio, et al., “High-Throughput RAD-SNP Genotyping forCharacterization of Sugar Watermelon Genotypes” Biotechnology PlantMolecular Biology Reporter (January 2013) 32(3) and Schneider, K., etal., “Analysis of DNA polymorphisms in sugar beet (Beta vulgaris L.) anddevelopment of an SNP-based map of expressed genes” Theor Appl Genet.(September 2007); 115(5):601-15.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome. See for example, Gildner, S., et al., “QTL mapping of BNYVVresistance from the WB41 source in sugar beet” Genome. (April 2005)48(2):279-285 and Setiawan, A., et al., “Mapping quantitative trait loci(QTLs) for resistance to Cercospora leaf spot disease (Cercosporabeticola Sacc.) in sugar beet (Beta vulgaris L.)”, (June 2000)Theoretical and Applied Genetics 100(8):1176-1182. QTL markers can alsobe used during the breeding process for the selection of qualitativetraits. For example, markers closely linked to alleles or markerscontaining sequences within the actual alleles of interest can be usedto select plants that contain the alleles of interest during abackcrossing breeding program. The markers can also be used to selectfor the genome of the recurrent parent and against the genome of thedonor parent. Using this procedure can minimize the amount of genomefrom the donor parent that remains in the selected plants. It can alsobe used to reduce the number of crosses back to the recurrent parentneeded in a backcrossing program. The use of molecular markers in theselection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Expression Vectors for Watermelon Transformation: Marker Genes

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). Expression vectors includeat least one genetic marker operably linked to a regulatory element (forexample, a promoter) that allows transformed cells containing the markerto be either recovered by negative selection, i.e., inhibiting growth ofcells that do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well-known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or an herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin.

Additional selectable marker genes include Pain1-9a and Pain1-8c whichboth correspond to the group a alleles of the vacuolar acid invertasegene; Pain1prom-d/e; Stp23-8b, StpL-3b, and StpL-3e which originate fromtwo plastid starch phosphorylase genes; AGPsS-9a which is positivelyassociated an increase in plant starch content, starch yield and chipquality, and AGPsS-10a which is associated with a decrease in theaverage plant starch content, starch yield and chip quality; GP171-awhich corresponds to allele 1a of ribulose bisphosphate carboxylaseactivase; and Rca-1a.

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase (Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); Charest, et al., Plant Cell Rep.,8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells, rather than directgenetic selection of transformed cells, for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used marker genes forscreening presumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., Proc. Natl. Acad. Sci. USA, 84:131(1987); DeBlock, et al., EMBO J., 3:1681 (1984)).

Expression Vectors for Watermelon Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell-type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions. Many types of promoters are well known in theart.

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment, such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.Many signal sequences are well-known in the art. See, for example,Becker, et al., Plant Mol. Biol., 20:49 (1992); Knox, C., et al., PlantMol. Biol., 9:3-17 (1987); Lerner, et al., Plant Physiol., 91:124-129(1989); Frontes, et al., Plant Cell, 3:483-496 (1991); Matsuoka, et al.,Proc. Natl. Acad. Sci., 88:834 (1991); Gould, et al., J. Cell. Biol.,108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991); Kalderon, etal., Cell, 39:499-509 (1984); Steifel, et al., Plant Cell, 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes: Transformation

With transgenic plants according to one embodiment, a foreign proteincan be produced in commercial quantities. Thus, techniques for theselection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein can then beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to an embodiment, the transgenic plant provided for commercialproduction of foreign protein is a watermelon plant. In anotherembodiment, the biomass of interest is seed.

For the relatively small number of transgenic plants that show higherlevels of expression, a genetic map can be generated, primarily viaconventional RFLP, PCR, and SSR analysis, which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see, Glick and Thompson, Methodsin Plant Molecular Biology and Biotechnology, CRC Press, Inc., BocaRaton, 269:284 (1993). Map information concerning chromosomal locationis useful for proprietary protection of a subject transgenic plant.

Likewise, by means of one embodiment, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of watermelon, the expression of genes can be alteredto enhance disease tolerance, insect tolerance, herbicide tolerance,agronomic, grain quality, and other traits. Transformation can also beused to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to watermelons, as well asnon-native DNA sequences, can be transformed into watermelons and usedto alter levels of native or non-native proteins. Various promoters,targeting sequences, enhancing sequences, and other DNA sequences can beinserted into the genome for the purpose of altering the expression ofproteins. The interruption or suppression of the expression of a gene atthe level of transcription or translation (also known as gene silencingor gene suppression) is desirable for several aspects of geneticengineering in plants.

Many techniques for gene silencing are well-known to one of skill in theart, including, but not limited to, knock-outs (such as by insertion ofa transposable element such as Mu (Vicki Chandler, The Maize Handbook,Ch. 118 (Springer-Verlag 1994)) or other genetic elements such as a FRT,Lox, or other site specific integration sites; antisense technology(see, e.g., Sheehy, et al., PNAS USA, 85:8805-8809 (1988) and U.S. Pat.Nos. 5,107,065, 5,453,566, and 5,759,829); co-suppression (e.g., Taylor,Plant Cell, 9:1245 (1997); Jorgensen, Trends Biotech., 8(12):340-344(1990); Flavell, PNAS USA, 91:3490-3496 (1994); Finnegan, et al.,Bio/Technology, 12:883-888 (1994); Neuhuber, et al., Mol. Gen. Genet.,244:230-241 (1994)); RNA interference (Napoli, et al., Plant Cell,2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp, Genes Dev., 13:139-141(1999); Zamore, et al., Cell, 101:25-33 (2000); Montgomery, et al., PNASUSA, 95:15502-15507 (1998)), virus-induced gene silencing (Burton, etal., Plant Cell, 12:691-705 (2000); Baulcombe, Curr. Op. Plant Bio.,2:109-113 (1999)); target-RNA-specific ribozymes (Haseloff, et al.,Nature, 334:585-591 (1988)); hairpin structures (Smith, et al., Nature,407:319-320 (2000); U.S. Pat. Nos. 6,423,885, 7,138,565, 6,753,139, and7,713,715); MicroRNA (Aukerman & Sakai, Plant Cell, 15:2730-2741(2003)); ribozymes (Steinecke, et al., EMBO J., 11:1525 (1992);Perriman, et al., Antisense Res. Dev., 3:253 (1993)); oligonucleotidemediated targeted modification (e.g., U.S. Pat. Nos. 6,528,700 and6,911,575); Zn-finger targeted molecules (e.g., U.S. Pat. Nos.7,151,201, 6,453,242, 6,785,613, 7,177,766 and 7,788,044); and othermethods or combinations of the above methods known to those of skill inthe art.

The foregoing methods for transformation may be used for producing atransgenic variety. The transgenic variety could then be crossed withanother (non-transformed or transformed) variety in order to produce anew transgenic variety. Alternatively, a genetic trait that has beenengineered into a particular watermelon line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties that do not contain that gene. As used herein,“crossing” can refer to a simple x by y cross or the process ofbackcrossing depending on the context.

Likewise, by means of one embodiment, agronomic genes can be expressedin transformed plants. More particularly, plants can be geneticallyengineered to express various phenotypes of agronomic interest,including, but not limited to, genes that confer resistance or toleranceto pests or disease, genes that confer resistance or tolerance to anherbicide, genes that confer or contribute to a value-added or desiredtrait, genes that control male sterility, genes that create a site forsite specific DNA integration, and genes that affect abiotic stresstolerance. Many hundreds if not thousands of different genes are knownand could potentially be introduced into a watermelon plant according tothe invention. Non-limiting examples of particular genes andcorresponding phenotypes one may choose to introduce into a watermelonplant include one or more genes for insect tolerance, such as a Bacillusthuringiensis (B.t.) gene, pest tolerance such as genes for fungaldisease control, herbicide tolerance such as genes conferring glyphosatetolerance, and genes for quality improvements such as yield, nutritionalenhancements, environmental or stress tolerances, or any desirablechanges in plant physiology, growth, development, morphology or plantproduct(s). For example, structural genes would include any gene thatconfers insect tolerance including but not limited to a Bacillus insectcontrol protein gene as described in WO 99/31248, herein incorporated byreference in its entirety, U.S. Pat. No. 5,689,052, herein incorporatedby reference in its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275,herein incorporated by reference in their entirety. In anotherembodiment, the structural gene can confer tolerance to the herbicideglyphosate as conferred by genes including, but not limited toAgrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA:CP4) asdescribed in U.S. Pat. No. 5,633,435, herein incorporated by referencein its entirety, or glyphosate oxidoreductase gene (GOX) as described inU.S. Pat. No. 5,463,175, herein incorporated by reference in itsentirety. Alternatively, the DNA coding sequences can affect thesephenotypes by encoding a non-translatable RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillito, Mol.Biotech., 7:125, 1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of one or more embodiments.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of watermelons andregeneration of plants therefrom is well-known and widely published.See, for example, Xu, Q. L., et al., “Efficient plant regeneration invitro from red leaf watermelon via organogenesis”, Russian Journal ofPlant Physiology (2009) 56: 546-550 and Mezei, S., et al., “SugarWatermelon Micropropagation”, (April 2014) Biotechnology &Biotechnological Equipment. 20(1). Thus, another aspect or embodiment isto provide cells which upon growth and differentiation producewatermelon plants having the physiological and morphologicalcharacteristics of inbred watermelon line WTL0088, WTL0089, WDL0090 orWTL0091.

Regeneration refers to the development of a plant from tissue culture.The term “tissue culture” indicates a composition comprising isolatedcells of the same or a different type or a collection of such cellsorganized into parts of a plant. Exemplary types of tissue cultures areprotoplasts, calli, plant clumps, and plant cells that can generatetissue culture that are intact in plants or parts of plants, such asembryos, root, pollen, flowers, seeds, pods, petioles, leaves, stems,roots, root tips, anthers, pistils, and the like. Means for preparingand maintaining plant tissue culture are well known in the art. By wayof example, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and 5,977,445describe certain techniques, the disclosures of which are incorporatedherein by reference.

INDUSTRIAL USES

Watermelon offers a wide variety of uses in for fresh, processed andcooked fruit. For example, watermelons can be eaten raw or cooked(fried, baked, boiled, etc). Watermelons can also be used to make foodcoloring, medicine, powders, juices, sweetener, colorings, watermelonpulp, watermelon shreds, raffinate and betaine. Thus, a furtherembodiment provides for a food product made from a part of thewatermelon plant variety WTL0088, WTL0089, WDL0090 or WTL0091.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

One embodiment may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

Various embodiments, include components, methods, processes, systemsand/or apparatus substantially as depicted and described herein,including various embodiments, sub-combinations, and subsets thereof.Those of skill in the art will understand how to make and use anembodiment(s) after understanding the present disclosure.

The foregoing discussion of the embodiments has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the embodiments to the form or forms disclosed herein. In theforegoing Detailed Description for example, various features of theembodiments are grouped together in one or more embodiments for thepurpose of streamlining the disclosure. This method of disclosure is notto be interpreted as reflecting an intention that the embodiment(s)requires more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this Detailed Description.

Moreover, though the description of the embodiments has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the embodiments (e.g., as may be within the skill and knowledge ofthose in the art, after understanding the present disclosure). It isintended to obtain rights which include alternative embodiments to theextent permitted, including alternate, interchangeable and/or equivalentstructures, functions, ranges or acts to those claimed, whether or notsuch alternate, interchangeable and/or equivalent structures, functions,ranges or acts are disclosed herein, and without intending to publiclydedicate any patentable subject matter.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the embodiments (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the embodiments unless otherwise claimed.

Deposit Information

A deposit of least 625 seeds of the Sakata Seed America, Inc.proprietary inbred watermelon line WTL0088 disclosed above and recitedin the appended claims has been made with the Provasoli-GuillardNational Center for Marine Algae and Microbiota, Bigelow Laboratory forOcean Sciences (NCMA), 60 Bigelow Drive, East Boothbay, Me. 04544. Thedate of deposit was Mar. 3, 2021. The NCMA No. is 202103002. The depositof plant tissue was taken from the same deposit maintained by SakataSeed America, Inc. since prior to the filing date of this application.The deposit will be maintained in the NCMA depository for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced ifnecessary, during that period. Upon issuance, all restrictions on theavailability to the public of the deposit will be irrevocably removedconsistent with all of the requirements of 37 C.F.R. §§ 1.801-1.809.

A deposit of least 625 seeds of the Sakata Seed America, Inc.proprietary inbred watermelon line WTL0089 disclosed above and recitedin the appended claims has been made with the Provasoli-GuillardNational Center for Marine Algae and Microbiota, Bigelow Laboratory forOcean Sciences (NCMA), 60 Bigelow Drive, East Boothbay, Me. 04544. Thedate of deposit was Mar. 3, 2021. The NCMA No. is 202103003. The depositof plant tissue was taken from the same deposit maintained by SakataSeed America, Inc. since prior to the filing date of this application.The deposit will be maintained in the NCMA depository for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced ifnecessary, during that period. Upon issuance, all restrictions on theavailability to the public of the deposit will be irrevocably removedconsistent with all of the requirements of 37 C.F.R. §§ 1.801-1.809.

A deposit of least 625 seeds of the Sakata Seed America, Inc.proprietary inbred watermelon line WDL0090 disclosed above and recitedin the appended claims has been made with the Provasoli-GuillardNational Center for Marine Algae and Microbiota, Bigelow Laboratory forOcean Sciences (NCMA), 60 Bigelow Drive, East Boothbay, Me. 04544. Thedate of deposit was Mar. 3, 2021. The NCMA No. is 202103004. The depositof plant tissue was taken from the same deposit maintained by SakataSeed America, Inc. since prior to the filing date of this application.The deposit will be maintained in the NCMA depository for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced ifnecessary, during that period. Upon issuance, all restrictions on theavailability to the public of the deposit will be irrevocably removedconsistent with all of the requirements of 37 C.F.R. §§ 1.801-1.809.

A deposit of least 625 seeds of the Sakata Seed America, Inc.proprietary inbred watermelon line WTL0091 disclosed above and recitedin the appended claims has been made with the Provasoli-GuillardNational Center for Marine Algae and Microbiota, Bigelow Laboratory forOcean Sciences (NCMA), 60 Bigelow Drive, East Boothbay, Me. 04544. Thedate of deposit was Mar. 3, 2021. The NCMA No. is 202103005. The depositof plant tissue was taken from the same deposit maintained by SakataSeed America, Inc. since prior to the filing date of this application.The deposit will be maintained in the NCMA depository for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced ifnecessary, during that period. Upon issuance, all restrictions on theavailability to the public of the deposit will be irrevocably removedconsistent with all of the requirements of 37 C.F.R. §§ 1.801-1.809.

What is claimed is:
 1. A seed of inbred watermelon line WDL0090, whereina representative sample of seed of said inbred watermelon line wasdeposited under NCMA No.
 202103004. 2. A watermelon plant, or a partthereof, produced by growing the seed of claim
 1. 3. A plant of inbredwatermelon line WDL0090, or a part thereof, wherein a representativesample of seed of said inbred watermelon line was deposited under NCMANo.
 202103004. 4. A tissue culture produced from protoplasts or cellsfrom the plant of claim 3, wherein said cells or protoplasts areproduced from a plant part selected from the group consisting of leaf,pollen, ovule, embryo, cotyledon, taproot, hypocotyl, meristematic cell,callus, root, root tip, pistil, anther, flower, fruit, seed, shoot,stem, and petiole.
 5. A watermelon plant regenerated from the tissueculture of claim 4, wherein said plant has all of the physiological andmorphological characteristics of inbred watermelon line WDL0090.
 6. Amethod of producing a progeny watermelon seed, wherein the methodcomprises crossing the plant of claim 3 with a different watermelonplant and harvesting the resultant watermelon seed.
 7. A watermelon seedproduced by the method of claim
 6. 8. A watermelon plant, produced bygrowing the seed of claim
 7. 9. A method of producing a progenywatermelon seed, wherein the method comprises selfing the plant of claim3 and harvesting the resultant watermelon seed.
 10. A watermelon seedproduced by the method of claim
 9. 11. A watermelon plant, or a partthereof, produced by growing the seed of claim
 10. 12. A method ofintroducing a desired trait into inbred watermelon line WDL0090, whereinthe method comprises: (a) crossing an inbred WDL0090 plant, wherein arepresentative sample of seed of inbred watermelon line WDL0090 wasdeposited under NCMA No. 202103004, with a plant of another inbredwatermelon line that comprises a desired trait to produce progenyplants; (b) selecting one or more progeny plants that have the desiredtrait; (c) backcrossing the selected progeny plants with inbred WDL0090plants to produce backcross progeny plants; (d) selecting for backcrossprogeny plants that have the desired trait; and (e) repeating steps (c)and (d) two or more times in succession to produce selected third orhigher backcross progeny plants that comprise the desired trait and allof the physiological and morphological characteristics of inbredwatermelon line WDL0090.
 13. A watermelon plant produced by the methodof claim 12 wherein said watermelon plant contains the desired trait andall of the physiological and morphological characteristics of watermelonWDL0090.
 14. The watermelon plant of claim 13, wherein the desired traitis selected from the group consisting of male sterility, herbicidetolerance, insect tolerance, pest tolerance, disease tolerance,environmental stress tolerance, and modified carbohydrate metabolism.15. A method for producing a seed of a watermelon plant derived frominbred watermelon line WDL0090 comprising the steps of: (a) crossing thewatermelon plant of claim 3 with itself or a second watermelon plant;and (b) allowing seed of an inbred WDL0090-derived watermelon plant toform.
 16. The method of claim 15, further comprising the steps of: (a)growing a plant from said inbred WDL0090-derived watermelon seed toyield additional inbred WDL0090-derived watermelon seed; and (b) growingsaid additional inbred WDL0090-derived watermelon seed of step (a) toyield additional inbred WDL0090-derived watermelon plants.
 17. Themethod of claim 15, wherein said second watermelon plant is an inbredwatermelon plant, wherein the method further comprises: sowing said seedfrom inbred WDL0090-derived watermelon plant; and growing saidwatermelon seed of claim 15 to yield a hybrid watermelon plant.
 18. Themethod of claim 15, wherein said second watermelon plant is chosen fromthe female inbred lines WTL0088 wherein a representative sample of seedof said inbred watermelon line was deposited under NCMA No. 202103002,WTL0089 wherein a representative sample of seed of said inbredwatermelon line was deposited under NCMA No. 202103002 and WTL0091wherein a representative sample of seed of said inbred watermelon linewas deposited under NCMA No.
 202103005. 19. The method of claim 18,wherein female inbred line WTL0088 is crossed with male inbred lineWDL0090 to produce hybrid watermelon line XWT8009, female inbred lineWTL0091 is crossed with male inbred line WDL0090 to produce hybridwatermelon line XWT8018, and female inbred line WTL0089 is crossed withmale inbred line WDL0090 to produce hybrid watermelon line XWT8028. 20.A method of vegetatively propagating a plant of inbred watermelon lineWDL0090 comprising the steps of: (a) collecting tissue capable of beingpropagated from the plant of claim 3; (b) cultivating said tissue toobtain proliferated shoots; and (c) rooting said proliferated shoots toobtain rooted plantlets.
 21. The method of claim 20, further comprisinggrowing plants from said rooted plantlets.
 22. A plant part of the plantof claim 3, wherein the plant part comprises a cell of said inbredwatermelon line WDL0090.
 23. A method for developing a watermelon plantin a plant breeding program, comprising applying plant breedingtechniques to the plant, or plant part thereof, of claim 3, wherein saidplant breeding techniques are selected from the group consisting ofcrossing, recurrent selection, mutation breeding, wherein said mutationbreeding selects for a mutation that is spontaneous or artificiallyinduced, backcrossing, pedigree breeding, marker enhanced selection,haploid/double haploid production, or transformation, and whereinapplication of said techniques results in development of a watermelonplant.
 24. A method of introducing a mutation into the genome of aninbred watermelon plant of line WDL0090, said method comprisingmutagenizing the plant, or plant part thereof, of claim 3, wherein saidmutagenizing is selected from the group consisting of temperature,long-term seed storage, tissue culture conditions, ionizing radiation,chemical mutagens, or targeting induced local lesions in genomes, andwherein the resulting plant comprises at least one genome mutation. 25.A method of editing a gene in the genome of inbred watermelon plant ofline WDL0090, said method comprises editing the genome of the plant, orplant part thereof, of claim 3, wherein said gene editing utilizes zincfinger nucleases, transcription activator-like effector nucleases(TALENs), engineered homing endonucleases/meganucleases, or theclustered regularly interspaced short palindromic repeat(CRISPR)-associated protein9 (Cas9) system.
 26. A watermelon plantproduced by the method of claim 25, wherein said plant has the editedgene and otherwise contains all of the morphological and physiologicalcharacteristics of watermelon plant WDL0090.
 27. The plant part of claim2, wherein said plant part is a fruit, a leaf, pollen, an ovule, a cell,a scion, a root, a rootstock, a cutting, or a flower.